The long range goal of this research is to clarify factors which regulate the protein folding mechanism. Carbonic anhydrase I from human erythrocytes will be used as the primary standard test protein. The highly sensitive technique of differential scanning calorimetry will be used to monitor conformational changes. The initial specific aims are: (1) to monitor thermal unfolding of the protein and to generate a thermodynamic data set of changes in enthalpy and heat capacity which characterize the thermal unfolding pathway; (2) to better understand factors which influence the protein's folding mechanism, we will examine the unfolding process under a variety of conditions of pH, organic co- solvents and metal ion replacement; and (3) in an attempt to evaluate the relationship between amino acid sequence and three-dimensional structure, we will monitor and compare the thermal unfolding of a closely related isozyme, carbonic anhydrase II. Human erythrocyte carbonic anhydrases I and II are genetically and immunologically distinct, have nearly identical molecular mass, share 60% sequence homology and contains a single zinc ion per molecule. The reversible folding of proteins is a physical property of fundamental importance. Clarification of the influence of solvent environment, amino acid sequence and metal ion ligands on the folding process will have a far-reaching impact in biological and medical sciences including protein engineering. The carbonic anhydrases represent model metalloproteins highly sutied to this analysis.